High-throughput screening carbon and nitrogen sources to promote growth and sporulation in Rhizopus arrhizus

Rhizopus arrhizus is a saprotrophic, sometimes clinically- and industrially-relevant mold (Mucorales) and distributed worldwide, suggesting it can assimilate a broad spectrum of substrates. Here, 69 strains of R. arrhizus were investigated by using the Biolog FF MicroPlate for the profiles of utilizing 95 carbon and nitrogen substrates. The study showed that most R. arrhizus strains were similar in average well color development (AWCD) and substrate richness (SR). Nevertheless, 13 strains were unique in principal component analyses, heatmap, AWCD, and SR analyses, which may imply a niche differentiation within R. arrhizus. The species R. arrhizus was able to utilize all the 95 carbon and nitrogen substrates, consistent with the hypothesis of a great metabolic diversity. It possessed a substrate preference of alcohols, and seven substrates were most frequently utilized, with N-acetyl-d-galactosamine and l-phenylalanine ranking at the top of the list. Eight substrates, especially l-arabinose and xylitol, were capable of promoting sporulation and being applied for rejuvenating degenerated strains. By phenotyping R. arrhizus strains in carbon and nitrogen assimilation capacity, this study revealed the extent of intra-specific variability and laid a foundation for estimating optimum substrates that may be useful for industrial applications. Supplementary Information The online version contains supplementary material available at 10.1186/s13568-024-01733-0.


Introduction
Rhizopus arrhizus (Fungi, Mucoromycota, Mucoromycetes, Mucorales, and Rhizopodaceae) was proposed by Alfred Fischer in 1892, synonymizing Rhizopus oryzae Went and Prins.Geerl.1895.The species is characterized by sparse rhizoids, apophysate sporangia containing small and regular sporangiospores, and sporangiophores forming on stolons at the same point as rhizoids but in opposite direction (Zheng et al. 2007;Zhao et al. 2023).The R. arrhizus species are classified into three varieties, namely var.arrhizus, var.tonkinensis, and var.delemar, based on morphological and molecular phylogenetic evidence (Zheng et al. 2007;Liu et al. 2008;Dolatabadi et al. 2014), which is supported by biochemistry and physiology (Londoño-Hernández et al. 2017;Saito et al. 2004;Yao et al. 2018).All these suggest that R. arrhizus has a phylogenetic and genetic complexity, and a morphological diversity.A comprehensive study was carried out to understand the pattern in intraspecific variation, but found no specific adaptations to environment (Kaerger et al. 2015).
R. arrhizus is found all over the world, colonizing a wide range of ecological habitats.Most strains of R. arrhizus are saprotrophic inhabitants of soil, dung, organic matter, and plant debris, while some isolates parasitize plants and infect animals and humans, causing mucormycosis, especially those co-infected with COVID-19 (Zheng et al. 2007;Kwon et al. 2011;Cheng et al. 2017;Ju et al. 2020;Tabarsi et al. 2021).The species plays an important role in industrial bio-transformations, such as beverage and food processing through fermentation (Jin et al. 2003;Liu et al. 2017;Dobrev et al. 2018).Meanwhile, R. arrhizus has a strong ability to uptake a wide range of substrates, including rice meal, soybean meal, glucose, and corn pulp, as feedstock to yield many productions that were widely used in food, therapeutic compounds and other biotechnological industries (Liu et al. 2017;Yao et al. 2018;López-Fernández et al. 2021;Bai et al. 2022;Corzo-León et al. 2023).
High-throughput phenotyping is a cornerstone of system biology, allowing simultaneous measurements of numerous taxa and characters (Cuevas and Edwards 2018).The Biolog automatic microbial analysis system, one of the high-throughput phenotypic assays, is able to identify organisms rapidly, effectively and accurately (Yao et al. 2006).This system, especially the FF MicroPlate designed particularly for filamentous fungi, is used to test the utilization of as many as 95 kinds of carbon/nitrogen sources at the same time (Tang et al. 2015;Schwendner and Schuerger 2018;Kabtani et al. 2024).For instance, the ability to metabolize most carbon/nitrogen sources was found to be lost at a pressure of 0.7 kPa in Sarrella liquefaciens (Schwendner and Schuerger 2018).Biolog system was also used to estimate resistance/sensitivity and metabolic activities against fungicides in filamentous fungi, such as R. arrhizus, Botrytis cinerea, and Fusarium spp.(Frąc et al. 2016;Wang et al. 2016Wang et al. , 2018)).Previous study showed that Biolog FF plates facilitated optimizing media for R. arrhizus and Rhizopus microsporus (Kordowska-Wiater et al. 2012).Closely related fungal strains were able to be discriminated using Biolog FF plates, e.g., those in Aspergillus spp., Coprinus comatus, Ganoderma lucidum, Oidiodendron fimicola, and Petriella setifera (Rice and Currah 2005;Singh 2009;Pawlik et al. 2015aPawlik et al. , 2015b;;Rola et al. 2015;Pertile et al. 2018).
In this study, we applied the Biolog FF MicroPlate to a diverse culture collection of the filamentous fungus R. arrhizus, assessing its diversity in carbon/nitrogen source assimilation capacity, screening for strains of potential use for industrial fermentations, as well as estimating optimum substrates for its growth and sporulation.

Strain collections
A total of 69 strains of R. arrhizus were collected from five continents covering 21 countries (Table 1).They were isolated from clinical, domesticated, and natural samples, such as skin, insects, flowers, koji, cakes, wrappers, air, and soil.Cultures were preserved at -80 °C in 15% glycerol at China General Microbiological Culture Collection Center (CGMCC, China), Westerdijk Fungal Biodiversity Institute (CBS, the Netherlands), Shandong Normal University (XY, China), and University of Michigan (UM, USA).And more information of 69 strains of R. arrhizus were listed in Table 1.

Morphological and phylogenetical analyses
The 69 strains collected were classified into three varieties: Rhizopus var.arrhizus, var.tonkinensis, and var.delemar (Table 1), based on a combination of morphology and DNA sequences targeting the ITS and IGS rDNA (Dolatabadi et al. 2014;Liu et al. 2008;Zheng et al. 2007).

Strain sporulation
Strains were revived on malt extract agar (MEA: malt extract 20 g/L, peptone 1 g/L, glucose 20 g/L, agar 20 g/L, pH 7.0), at 30 °C for a duration of 2 days.Aerial hyphae were then delicately scraped off using inverted 200 μL pipette tips, and 5 mm diameter inoculates were obtained by punching the inverted tip into the medium.Single inoculate was cultivated at 30 °C for 3 days in 250 mL flasks containing 20 mL of synthetic Mucor liquid (SML: glucose 20 g/L, asparagine 2 g/L, K 2 HPO 4 0.5 g/L, MgSO 4 ⋅7H 2 O 0.25 g/L, vitamin B 1 0.5 mg/L).For strains showing little or no sporangiospore productions, cultivation extended up to 20 days.Notably, five R. arrhizus strains (XY00507, XY01736, XY03794, XY03822, and XY03829, Table 1) were further examined due to their lack of sporulation in the SML medium.
In cases where strains did not produce sporangiospores, inoculation onto MEA solid media was employed to collect sporangiospores for subsequent suspension in filamentous fungus inoculating fluid (FF-IF) medium.Under a stereomicroscope (Leica, MDG33, Singapore), the Biolog FF MicroPlate was meticulously examined.
Each well was individually photographed after 7 days of growth to ensure sufficient sporulation.Sporangiospores from each well were eluted three times with 200 μL of sterile water and transferred into 1 mL Eppendorf (EP) tubes.Hemocytometer counts and microscopic observations (Zeiss, Axio Imager A2, Oberkohen, Germany) were employed to determine spore concentrations.Strains with spore concentrations of 0, 1.50E5 −5.25E5 , 8.75E5 −2.00E6 , and 2.50E6 −1.14E7 sporangiospores/mL were categorized as non-promoted, poorly promoted, moderately promoted, and heavily promoted strains, respectively.Substrate promotion frequency (PF) and high promotion frequency (HPF) denoted the proportions of promoted and heavily promoted strains among all five tested strains, respectively.

High-throughput screening carbon/nitrogen sources
In this study, we used well absorbance values, biological activities, strain substrate richness, and substrate utilization frequency to estimate carbon/nitrogen assimilations of R. arrhizus strains through FF MicroPlates containing 95 different carbon and nitrogen sources.
Strains were revived on solid media MEA at 30 °C for 4 days or until sporangia were abundantly produced.Sporangiospores were swabbed off the agar media using a sterile cotton swab pre-macerated with filamentous fungus inoculating fluid (FF-IF Biolog catalog #72,106, 0.25% Phytagel and 0.03% Tween 40 in DI water, Biolog Inc., Hayward, CA, USA), avoiding carryover nutrients from the agar medium, and then they were suspended into a 10 mL tube containing 5 mL of FF-IF.Suspensions were homogenized by adjusting to an optical density (OD) value of 0.15 at a wavelength of 590 nm with a 752 UV  S1) and calibrated by initial values.
The biological activity of the strains was quantified through an overall source utilization metric, which encompassed the calculation of the average well color development (AWCD, represented the average well color development data on the 4th day).The AWCD index was adjusted by subtracting values obtained from the blank (water) and calculated using the formula AWCD = [Σ(Ci − R)]/95, where 95 represented the number of wells or substrates, Ci denoted the optical density of substrate measured at 490 nm, and R represented the optical density of the control zero-carbon well (Garland and Mills 1991).To facilitate comparisons between different groups, a one-way MANOVA analysis focusing on AWCD was conducted for three varieties (var.arrhizus, var.delemar, and var.tonkinensis) and three origins (clinical, domesticated, and natural) of strains (Table 1).
When the difference between a well's absorbance and its control value (C-R) exceeded 0.25 or 1.50, the corresponding carbon/nitrogen source was classified as an efficient or highly efficient substrate, respectively (Zou et al. 2016).The substrate utilization frequency (UF) and substrate high utilization frequency (HUF) were defined as the proportions of strains utilizing efficient and highly efficient substrates, respectively.These proportions were calculated as N/69, where 69 represented the total number of tested strains, and N denoted the count of strains utilizing efficient or highly efficient substrates.For comparative analysis across different groups, a oneway MANOVA focusing on UF was conducted for two types (carbon and nitrogen) or six substrate categories (alcohols, amines, amino acids, carbohydrates, carboxylic acids/derivatives, and glycosides, as per the classification from the DrugBank database (https://www.drugbank.ca/,Wishart et al. 2018).

Data analyses
The principal component analysis (PCA) and visualization of substrate utilization data were conducted using the web tool ClustVis (https://biit.cs.ut.ee/clustvis/,Metsalu and Vilo 2015).As the AWCD demonstrated a gradual increase followed by a stabilization from the 4th day onwards, PCA analyses and fingerprint mapping were based on absorbance values recorded at the 4th day.These values, controlled by zero carbon/nitrogen conditions and calibrated with the initial measurement, served as the foundation for the analyses.
In the PCA analyses, strains were considered as annotations, rows were scaled with unit variance, and principal components were calculated through the default singular value decomposition (SVD) method with imputation implemented in ClustVis website (https://bit.cs.ut.ee/clustvis/, Metsalu and Vilo 2015).Prediction ellipses were drawn at a 95% confidence level, and the X-and Y-axes were constrained to share the same scale.For heatmap creation, rows and columns were both clustered using correlation distance with single-linkage criterium, two-color gradient, and unit variance scaling.
Venn diagrams were generated using the OriginPro 2020 (https://www.originlab.com/,Seifert 2014).The sporulation promotion frequency (PF), high promotion frequency (HPF), growth utilization frequency (UF), and high utilization frequency (HUF) data of substrates were imported into the software as sheets, and the range was specified based on respective values.The process involved selecting the "Venn Diagram" app implemented within OriginPro to create the graphical representation.Additionally, adjustments to the color and labeling of each set in the Venn diagram were made to align with the specific requirements of the data.
In addition, the statistical analysis was carried out using IBM SPSS Statistics for Windows, version 19.0 (IBM Corp., Armonk, N.Y., USA).

Carbon and nitrogen assimilation abilities of Rhizopus arrhizus
The absorbance values for the 69 R. arrhizus strains across 96 wells were carefully monitored and calibrated over a 7-day period.The average well color development (AWCD) exhibited a swift increase during the exponential phase from the beginning to the 4th day, and a nearly stable trend in the stationary phase from the 4th to the 7th day (Fig. 1A).
The principal component analysis (PCA, Fig. 2A) of 69 strains of R. arrhizus revealed that the primary sources of variability were captured by the two components (PC1 and PC2) based on the controlled and calibrated absorbance values on the 4th day.The majority of strains formed a cohesive cluster, showcasing similar patterns in carbon/nitrogen source utilization.While three strains, namely XY03787, XY03802, and XY03806, were outlier strains, indicating distinctive patterns.In summary, 13 strains were unique in PCA, heatmap, AWCD, and SR analyses, which may imply a niche differentiation within R. arrhizus (Supplemental Table S2).

Substrate preference of Rhizopus arrhizus
R. arrhizus has a preference for specific substrates, as demonstrated by variations in the average utilization frequency (UF) across six type substrates.A one-way MANOVA analysis revealed a significant difference (P < 0.01), with alcohols being the most preferred type (Fig. 2B).Notably, certain high-utilization substrates, particularly N-acetyl-d-galactosamine (A03) and l-phenylalanine (H03), with a UF > 95% and HUF > 75%, were identified as favorable for the growth of R. arrhizus (Fig. 4 and Supplemental Table S1).This substrate preference is instructive for the formulation and optimization of selective media (Pertile et al. 2018).
Beyond influencing the growth of R. arrhizus, various substrates were found to promote sporulation.When considering non-sporulation strains, only 50 out of the 95 substrates (53%) demonstrated the ability to induce sporulation (Fig. 4 and Supplemental Table S1).Among Fig. 2 Principal component analysis (PCA) and utilization frequency (UF) of Rhizopus arrhizus.A PCA of the 4th day's absorbance values for 69 strains of Rhizopus arrhizus while being incubated with Biolog FF MicroPlate.X and Y axes show PC1 and PC2 that explain 28.0 and 20.4% of the total variance, respectively.Three outliers are labeled as blue points and strain's codes, while other strains are only labeled as red points and encompassed with a 95% prediction ellipse.B The curve of utilization frequency (UF, Y-axis) of six categories of substrates by Rhizopus arrhizus during an incubation period of 7 days (X-axis).The highest efficient alcohols are significantly different from four categories (P = 2.28E −4 for carbohydrates, 4.81E −5 for amines, 9.58E −7 for glycosides, 6.15E −11 for carboxylic acids and derivates), except the amino acids (P = 0.14) the 95 substrates, l-arabinose (A09) and xylitol (E11) emerged as the most efficient substrates, meeting a combined criterion of UF > 80%, HUF > 10%, PF = 100%, and HPF = 60%.These substrates hold potential for rejuvenating degenerated strains that have lost the ability to sporulate (Supplemental Fig. S4).

Interspecific diversity analyses
Distinct variations in the fingerprints of carbon/nitrogen source metabolisms were evident in R. arrhizus (Fig. 5).While there were no clear boundaries separating carbon sources from nitrogen sources, the 95 carbon/nitrogen sources could be roughly categorized into two groups, as indicated by the two clades on the substrate phylogram (Fig. 5), which inconsistent with the morphology and molecular evidence (Zheng et al. 2007;Liu et al. 2008).Approximately 59% (56/95) of these sources exhibited low utilization, depicted in bluish tones and grouped on the left side of Fig. 5, whereas 41% (39/95) were highly utilized, represented in reddish tones and positioned on the right.
Fungal degeneration entails the spontaneous loss or decline in production capacity, resulting in significant economic losses.This phenomenon poses a threat Fig. 3 The macroscopic sporulation of XY03794 inoculated with the discrete carbon/nitrogen substrates fixed on the Biolog FF MicroPlate with 8 rows (A-H) and 12 columns (1-12).The detail of the carbon/nitrogen substrates refers to Supplemental Table S1 Fig. 4 The capability of 95 carbon/nitrogen substrates for promoting growth and sporulation.The bar chart on the left shows the accumulative percentage of sporulation promotion frequency (PF), high promotion frequency (HPF), growth utilization frequency (UF) and high utilization frequency (HUF).The Venn diagram on the upper right indicates the relationship between the strains with a UF > 95% or HUF > 75%, and that on the lower right displays those with a UF > 80%, HUF > 10%, PF = 100% or HPF = 60%.The red stars highlight the best substrates also revealed in relevant Venn diagrams, two for growth (A03: N-acetyl-d-galactosamine and H03: l-phenylalanine) and two for sporulation (A09: l-arabinose and E11: Xylitol).The detail of carbon/nitrogen refers to Supplemental Table S1 to some of the widely utilized fungal genera in the biotechnical industry, such as Aspergillus, Penicillium, and Trichoderma (Adrio and Demain 2003;Douma et al. 2011;Song et al. 2022;Danner et al. 2023).Similar with these fungi, as the number of passages increases and artificial domestication of fermentation environment is prolonged, certain R. arrhizus strains may progressively undergo degradation, which is primarily characterized by a sluggish expansion of mycelia, a reduction in sporangiospore quantities, and even a complete absence of sporangiospore production.The degradation of R. arrhizus strains will also hinder the application in food and industry.Previous studies suggest that strains can be reduced through various pathways, such as preservation methods, strains selection, and bioprocess design (Al-Bedak et al. 2019;Meyer et al. 2021;Danner et al. 2023).Our study shows that eight substrates can significantly promote the reproduction of sporangiospores in some strains of R. arrhizus, which cannot sporulate even after an extended incubation period of 20 days with the synthetic Mucor liquid (SML).
In addition, this study adopts a quick and easy way to obtain the metabolic profile of various carbon/nitrogen sources, which was illustrated by Yao et al. (2018).Single strain's metabolic profile reflects an individual metabolic ability, and a profile of a biota represents a population capacity.The metabolic profile of a fermentative biota could be used to monitor microbial quality and fermentation process (Prihatna and Suwanto 2007).Thanks to the ability to produce a variety of biological enzymes and metabolically active substances, Rhizopus is widely used in the food fermentation industry.R. arrhizus has a high Fig. 5 The clustered heatmap of the absorbance values at the 4th day for 69 strains of Rhizopus arrhizus (row) against 95 substrates (column) showing inter-strain and inter-substrate variations.The trees on the left and top illustrate clustering of the strains and substrates, respectively.Annotations about the carbon/nitrogen substrates are labeled on the top and their detail refers to Supplemental Table S1 utilization rate of carbohydrates (Fig. 4), in accordance with its well-known high saccharification ability (Yao et al. 2018).By tracking the change of metabolic fingerprints, the fermentation process and the biological activity of R. arrhizus could be under control.
The present study on the metabolic fingerprint showed that R. arrhizus was diverse in carbon/nitrogen assimilation and its comparative genomics, population genetics and epigenomics should be further investigated.Chibucos et al. (2016) have performed an integrated genomic and transcriptomic study on pathogenic Mucorales, including R. arrhizus, however, they did not take account of natural and industrial-relevant isolates.Previous reports on the metabolism of carbon/nitrogen sources in R. arrhizus rarely focused on its population diversity (Ghosh and Ray 2014), although Prihatna and Suwanto (2007) found a distinct separation in metabolic fingerprints among geographical origins of Rhizopus oligosporus, a species related to R. arrhizus.In the present study, we observed variation of substrate utilization within R. arrhizus, however, these distinctions did neither fall along the lines of isolation source (ecology) nor genetic lineage (taxonomic varieties), suggesting that divergence in gross substrate utilization has not evolved rapidly.At present, R. arrhizus were divided into three varieties, namely var.arrhizus, var.tonkinensis, and var.delemar (Zheng et al. 2007;Liu et al. 2008;Dolatabadi et al. 2014), and supported by some biochemistry and physiology studies (Londoño-Hernández et al. 2017;Saito et al. 2004;Yao et al. 2018).While two groups were categorized based on high utilization frequencies (HUFs), viz., 0-39.66% and 54.47-84.99%groups, which suggested R. arrhizus may have instead evolved to utilize a very broad and stable spectrum of carbon and nitrogen substrates.
This study identified several substrates that significantly promote the growth and sporangiospore production of R. arrhizus strains through high-throughput screening based on the Biolog FF MicroPlates, which containing 95 different carbon and nitrogen sources.The assimilation capabilities of R. arrhizus strains for different carbon and nitrogen sources were evaluated, revealing a certain substrate preference.Analyzing the assimilation capabilities of various R. arrhizus strains for carbon and nitrogen sources could be considered as a fingerprint for intra-specific variation.In summary, this study lays the foundation for the applications of R. arrhizus strains in fermentation and food industry, as well as provides new cultivation strategies to address strain degeneration.

Fig. 1
Fig. 1 Average well color development (AWCD, y-axis) against incubation time (x-axis).A The trend for 69 strains of R. arrhizus showing their carbon/nitrogen assimilation abilities while being incubated with Biolog FF MicroPlate for 7 days.AWCD values are means (standard deviations not shown) from three independent technical repeats each being read in triplicates.B The carbon/nitrogen assimilation trend for three varieties (var.arrhizus, var.tonkinensis, and var.delemar) of R. arrhizus.C The carbon/nitrogen assimilation trend for three origins (clinical, domesticated, and natural) of R. arrhizus